Staphylococcus spp. inhabiting backyard rabbits

Staphylococcus spp. inhabiting backyard rabbits

Staphylococcus spp. inhabiting backyard rabbits
Czech Collection of Microorganisms
http://www.sci.muni.cz/ccm/
1,*
2
2
3
Pavel Švec, Oto Melter, Jan Tkadlec, and Roman Pantůček
1
Czech Collection of Microorganisms, Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic
2
Department of Medical Microbiology, 2nd Faculty of Medicine, Charles University, Prague, Czech Republic
3
Division of Genetics and Molecular Biology, Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic
*
Corresponding author: [email protected]
Introduction
The genus Staphylococcus represents ubiquitous microorganisms
occurring mainly as common commensals of mammal's skin and mucous
membranes and individual species of the genus has been isolated from a
wide variety of different animal species. In contrast, staphylococci
represent important microorganisms involved in numerous human and
animal infections (Schleifer and Bell, 2009).
Breeding of backyard rabbits for meat has a long tradition and is
popular mainly in rural regions of Slovakia. However, there are nearly no
data about the natural microflora of rabbits raised by these small farmers.
Due to this reason we conducted this short study to analyse the taxonomic
structure of Staphylococcus spp. inhabiting the backyard rabbits.
Material and methods
Bacterial strains
A group of 64 staphylococcal strains was collected from clinically healthy
backyard rabbits kept in hutches by a small breeder in Spišská Nová Ves (Slovakia).
Analysed strains were isolated from nose (30 strains), ear (24), anal (6), and throat
(4) swab samples obtained from 16 weaned or adult rabbits. Individual swab
samples were cultivated on nutrient agar incubated overnight in microaerophilic
condition. White or yellow pigmented suspected colonies were picked up and
purified. Microscopy of all the suspected cultures revealing Gram-positive regular
cocci in clusters were further analysed.
Strain numbers indicate origin of strains according to the following code: 1-5
(hutch number) / A-F (rabbit) / K, U, Z or N (body part) / 1-3 (isolate number).
Individual body parts are labelled as follows: K, throat; U, ear; Z, anus; N, nose.
Reference strains were obtained from the Czech Collection of Microorganisms (CCM,
www.sci.muni.cz/ccm/).
Results
All but one (GTG)5-PCR fingerprints matched reference database entries and
assigned 36 strains as Staphylococcus vitulinus, 20 as Staphylococcus xylosus, four
as Staphylococcus succinus and remaining strains as single representatives of
Staphylococcus sciuri, Staphylococcus equorum and Staphylococcus haemolyticus.
Only strain 4BN2 did not match any reference fingerprint and was not identified
using this method (Fig. 1).
Sequencing of the rpoB gene identified strain 4BN2 as S. xylosus and confirmed
strain 1Z1 as S. sciuri.
MALDI TOF mass spectrometry failed to identify 22 S. vitulinus, 6 S. xylosus, 3 S.
succinus and a S. haemolyticus strain (Table 1).
Biotyping results obtained with STAPHYtest 16 commercial kit assigned correctly
only 20 S. xylosus isolates; remaining strains were not identified or were identified
erroneously (Table 1). All strains were clumping factor, coagulase and hyaluronidase
negative. Strain S. haemolyticus 2BK was susceptible to novobiocin, remaining
strains revealing inhibition zones  16 mm were found to be resistant.
Fig 1. Dendrogram based on cluster analysis of
(GTG)5-PCR fingerprints.
Similarity (%)
20
40
60
80
100
1Z1
P539
S. sciuri
2BK
CCM 2574 S. haemolyticus
5EU2
CCM 2210 S. equorum subsp. equorum
4BN2
2AN2
3CU2
CCM 4822
5CN2
5FU2
2CN2
5EN1
5DU2
5BU2
5CK
S. xylosus
1N1
1Z2
5EN2
5CN1
2BN
3BN1
4BU2
4CN2
2CZ
5CU2
4AN1
CCM 2738T
3CN2
3CN1
4AN3
4BU1
4BN1
CCM 4511T
3BU1
5FU1
5CU1
5BU1
2CU
3AU1
5DU1
3AN1
3CU1
5FN1
1U1
5DK
4CN1
S. vitulinus
2AU1
2BZ
3CZ1
5FN2
5DN1
1N2
5EU1
3AN2
3BN2
5DN2
CCM 4481
5AN
1U2
2AU2
2BU
3CZ2
1K1
4AU1
3AU2
CCM 4512
2CN1
2AN1
S. succinus
CCM 2495
4AN2
5BN
Repetitive extragenic palindromic-PCR with the (GTG)5 primer
(GTG)5-PCR fingerprinting, numerical analysis of the resulting fingerprints using
the Bionumerics 6.6 software (Applied-Maths, Belgium) and their comparison with
reference entries representing all known Staphylococcus spp. was performed as
described by Švec et al. (2010). The dendrogram was constructed using the
Pearson's correlation coefficients with the unweighted pair-group method using
arithmetic averages (UPGMA) clustering method.
MALDI-TOF mass spectrometry analysis
Isolated strains were analyzed using the Matrix-Assisted Laser Desorption/
Ionization Time-of-Flight Mass Spectrometry on an Ultraflex III instrument (Bruker
Daltonik) according to a standard sample preparation protocol of Bruker Daltonik
(Freiwald and Sauer, 2009). MALDI-TOF mass spectra were subjected to a numerical
analysis and the strains were identified according to a comparison with reference
MALDI-TOF mass spectra from Bruker database using the IVD MALDI BioTyper 2.2
software (Bruker Daltonik).
Biotyping
All isolates were tested using the STAPHYtest 16 (Erba Lachema, Czech
Republic). Clumping factor, coagulase and hyaluronidase production, and
novobiocin susceptibility were tested using Itest commercial tests (Itest, Czech
Republic).
References
Mellmann, A., Becker, K., von Eiff, C., Keckevoet, U., Schumann, P., Harmsen, D. (2006) Sequencing and staphylococci identification. Emerg Infect
Dis, 12, 333-336.
Schleifer, K. H. & Bell, J. A. (2009). Genus I. Staphylococcus Rosenbach 1884, 18AL (Nom. Cons. Opin. 17 Jud. Comm. 1958, 153.). In Bergey's
Manual of Systematic Bacteriology, The Firmicutes, vol. 3, pp. 392-421. Edited by P. De Vos, G. M. Garrity, D. Jones, N. R. Krieg, W. Ludwig, F. A. Rainey, K.
H. Schleifer & W. B. Whitman. New York: Springer.
Švec, P., Pantůček, R., Petráš, P., Sedláček, I. & Nováková, D. (2010). Identification of Staphylococcus spp. using (GTG)5-PCR fingerprinting. Syst
Appl Microbiol 33, 451-456.
Acknowledgements
200
(bp)
300
400
500
1000
1500
2000
2500
3000
4000
Molecular Weight Marker
5000
rpoB gene sequencing
Partial rpoB gene sequencing was performed according to Mellmann et al. (2006)
in the Eurofins MWG Operon sequencing facility (Ebersberg, Germany).
Table 1. Identification results obtained using
(GTG)5-PCR, MALDI TOF MS and STAPHYtest 16.
Strain
(GTG)5-PCR
MALDI TOF MS
STAPHYtest 16
1Z1
S. sciuri
S. sciuri
S. sciuri/lentus
5EU2
S. equorum
S. equorum
S. xylosus
2BK
S. haemolyticus
NI
S. auricularis
2AN1
S. succinus
S. xylosus
S. xylosus
2CN1
S. succinus
NI
S. xylosus
4AN2
S. succinus
NI
S. xylosus
5BN
S. succinus
S. succinus
NI
1K1
S. vitulinus
NI
S. sciuri/lentus
1N2
S. vitulinus
NI
S. sciuri/lentus
1U1
S. vitulinus
NI
S. capitis subsp. capitis
1U2
S. vitulinus
NI
NI
2AU1
S. vitulinus
NI
NI
2AU2
S. vitulinus
NI
NI
2BU
S. vitulinus
NI
NI
2BZ
S. vitulinus
NI
S. capitis subsp. capitis
2CU
S. vitulinus
NI
S. sciuri/lentus
3AN1
S. vitulinus
NI
S. sciuri/lentus
3AN2
S. vitulinus
S. vitulinus
S. sciuri/lentus
3AU1
S. vitulinus
S. vitulinus
S. sciuri/lentus
3AU2
S. vitulinus
NI
S. sciuri/lentus
3BN2
S. vitulinus
NI
NI
3BU1
S. vitulinus
NI
S. sciuri/lentus
3CN1
S. vitulinus
S. vitulinus
S. sciuri/lentus
3CN2
S. vitulinus
S. vitulinus
S. sciuri/lentus
3CU1
S. vitulinus
S. vitulinus
S. sciuri/lentus
3CZ1
S. vitulinus
NI
S. sciuri/lentus
3CZ2
S. vitulinus
NI
NI
4AN3
S. vitulinus
S. sciuri/lentus
S. vitulinus
4AU1
S. vitulinus
NI
S. sciuri/lentus
4BN1
S. vitulinus
S. vitulinus
S. sciuri/lentus
4BU1
S. vitulinus
NI
NI
4CN1
S. vitulinus
NI
S. sciuri/lentus
5AN
S. vitulinus
NI
NI
5BU1
S. vitulinus
NI
S. sciuri/lentus
5CU1
S. vitulinus
S. vitulinus
S. sciuri/lentus
5DK
S. vitulinus
S. vitulinus
S. sciuri/lentus
5DN1
S. vitulinus
S. vitulinus
S. sciuri/lentus
5DN2
S. vitulinus
S. vitulinus
S. auricularis
5DU1
S. vitulinus
NI
S. auricularis
5EU1
S. vitulinus
S. vitulinus
S. sciuri/lentus
5FN1
S. vitulinus
S. vitulinus
S. sciuri/lentus
5FN2
S. vitulinus
NI
S. sciuri/lentus
5FU1
S. vitulinus
S. vitulinus
S. sciuri/lentus
1N1
S. xylosus
S. xylosus
S. xylosus
1Z2
S. xylosus
NI
S. xylosus
2AN2
S. xylosus
NI
S. xylosus
2BN
S. xylosus
S. xylosus
S. xylosus
2CN2
S. xylosus
NI
S. xylosus
2CZ
S. xylosus
NI
S. xylosus
3BN1
S. xylosus
S. xylosus
S. xylosus
3CU2
S. xylosus
S. xylosus
S. xylosus
4AN1
S. xylosus
S. xylosus
S. xylosus
4BN2
S. xylosus
S. xylosus
S. xylosus
4BU2
S. xylosus
S. xylosus
S. xylosus
4CN2
S. xylosus
S. xylosus
S. xylosus
5BU2
S. xylosus
NI
S. xylosus
5CK
S. xylosus
S. xylosus
S. xylosus
5CN1
S. xylosus
S. xylosus
S. xylosus
5CN2
S. xylosus
S. xylosus
S. xylosus
5CU2
S. xylosus
S. xylosus
S. xylosus
5DU2
S. xylosus
S. xylosus
S. xylosus
5EN1
S. xylosus
S. xylosus
S. intermedius
5EN2
S. xylosus
S. xylosus
S. xylosus
5FU2
S. xylosus
NI
S. xylosus
NI, not identified
Conclusions
S. vitulinus and S. xylosus species are common inhabitants of healthy
rabbits while S. succinus, S. sciuri, S. equorum and S. haemolyticus were
less abundant species.
This study was supported by project CEB (CZ.1.07/2.3.00/20.0183).
XXXII Annual Meeting of the European Culture Collections' Organization
“Biodiversity: Sustainability vs. Regulations”
12-14 June, 2013, Athens, Greece
Individual animals were inhabited by one to three staphylococcal
species. All four S. succinus strains were isolated only from nose samples;
however no clear correlation between the species composition and the
body part swabbed was found.